pp: A newly identified CD45-associated protein

ABSTRACT

This invention relates to a newly identified protein useful for treating diseases of the immune system, methods for obtaining said protein, isolated nucleic acids encoding said protein, and methods for obtaining inhibitors of said protein. The protein of this invention is characterized by an apparent molecular weight of about 32 kD, an isoelectric point of about 4.0-4.5 and coprecipitation with CD45. The protein may also be used in in vitro or in vivo assays to identify inhibitors of T cell activation.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 08/004,199,entitled "pp32:A Newly Identified CD45-Associated Protein", filed Jan.13, 1993, now abandoned, which is a file wrapper-continuation of U.S.Ser. No. 07/688,019, also entitled "pp32:A Newly IdentifiedCD45-Associated Protein", filed. Apr. 19, 1991, now abandoned, both ofwhich are hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates to a protein isolated from human T lymphocytes,methods for obtaining said protein and uses thereof an isolated nucleicacid molecule encoding said protein and uses thereof, and methods forobtaining inhibitors for said protein and uses thereof.

BACKGROUND OF THE INVENTION

Resting T lymphocytes can be activated in vitro by monoclonal antibodiesagainst the T cell receptor complex (TCR-CD3) or against CD2, a 50kDglycoprotein. Activation of resting T lymphocytes by means of monoclonalantibodies leads to proliferation and differentiation and thereforemimics the action of the naturally occurring ligands for those receptors(antigen for the T cell receptor or the LFA-3 for CD2). The earlieststep of T cell activation by either monoclonal antibodies or the naturalligands is a phosphorylation of a limited number of intracellular andtransmembrane proteins (e.g. CD3 epsilon, CD3 zeta, CD4, CD8, CD45).Phosphorylation of proteins is thought to be mediated by intracellularprotein kinases which are activated upon the binding of monoclonalantibodies or the appropriate ligands and which phosphorylate proteinseither on tyrosine residues (protein tyrosine kinases) or on threonine-and/or serine residues (serine/threonine kinases). Alternatively, it ispossible that constitutive dephosphorylation could be inhibited by Tcell activation and could therefore be responsible for the increasedabundance of phosphoproteins observed. During the last ten years, anincreasing number of protein kinases has been identified, but only avery limited number of those are tyrosine kinases. These proteintyrosine kinases can be divided into two groups:

a) Tyrosine kinases that are also integral membrane proteins withextracellular ligand binding domains and intracellular catalytic domains(e.g. EGF receptor, PDGF receptor, insulin receptor); and

b) Tyrosine kinases that do not possess an extracellular domain ormembrane spanning region but associate with the inner leaf of the plasmamembrane (e.g. p56^(lck), p60^(src), p59^(fyn)).

Protein tyrosine kinases of both groups are encoded by protooncogenesand may therefore play a role in the origin of malignant cell growth.Recent studies have shown that protein tyrosine kinases play a key rolein the regulation of cell growth and differentiation. Tyrosine kinasescan themselves be phosphorylated on serine, threonine and tyrosine.Recent studies have shown that the enzymatic activity of certaintyrosine kinases is partially dependent upon the degree ofphosphorylation on tyrosine. This leads us to the conclusion that theactivity of tyrosine kinases is at least partially regulated by tyrosinephosphatases.

The CD45 molecule, an integral membrane tyrosine phosphatase, isexpressed on all hematopoietic cells, and seems to play a very importantregulatory role during an immune response. This is shown by a number orT cell functions that are either increased or inhibited by monoclonalantibodies against the CD45 protein. However, it is not known whetherthose antibodies modulate the enzymatic activity of the molecule orwhich proteins are the natural substrates for CD45. p56^(lck), a T cellspecific tyrosine kinase that is associated with CD4, has been discussedas a possible substrate for CD45. However, no biochemical data arecurrently available that directly prove an association between CD45 andp56^(lck). Therefore, no substrates for CD45 have been definitivelyidentified.

Using unconventional immunoprecipitation techniques we have identified apotential substrate of CD45 which is the subject of this patent. Themolecule is an intracellular protein with a relative apparent molecularweight of 32kD (SDS-PAGE) and a pI of 4.0 to 4.5. In resting T cells,this protein, "pp32", is constitutively phosphorylated on serine.Immunoprecipitation experiments with anti-CD45 monoclonal antibodieshave shown that pp32 is specifically associated with CD45. Besides pp32,a tyrosine kinase coprecipitates with the CD45 molecule. Thecoprecipitated protein kinase is responsible for in vitrophosphorylation of pp32 on tyrosine residues. The tyrosine kinase hasbeen identified by immunoprecipitation and subsequent peptide analysisas p56^(lck). The ability of CD45 to use tyrosine phosphorylated pp32 asa substrate in vitro provides further evidence that an enzyme-substraterelationship exists for the two molecules in vivo. Detailedelectrophoretic analysis or pp32 has subsequently shown that thisprotein exists (in resting T cells) in two isoforms (pp32 high and pp32low). Both isoforms show rapid changes during the activation of Tlymphocytes. The changes take place within 5 minutes after stimulationof resting T lymphocytes with monoclonaI antibodies specific for CD2 orwith Phorbol esters.

Based upon the amino acid sequences of peptide fragments of the isolatedpp32 protein, a nucleic acid molecule encoding pp32 has been isolated.

SUMMARY OF THE INVENTION

The newly identified protein of this invention is characterized by (a)an apparent molecular weight of about 32 kD as determined by SDS-PAGEanalysis, (b) an isoelectric point of about 4.0 to 4.5, and (c)coprecipitation with CD45. Various isoforms or derivatives of pp32 havealso been identified, including pp32 phosphorylated on serine (inresting T cells). The invention provides an isolated pp32 protein. Theprotein may be phosphorylated or may be unphosphorylated. In oneembodiment, the protein has an amino acid sequence shown in SEQ ID NO:2.

The invention further provides an isolated nucleic acid moleculeencoding a pp32 protein which can associate with CD45. In oneembodiment, the nucleic acid molecule has a nucleotide sequence shown inSEQ ID NO: 1. Other aspects of the invention include recombinantexpression vectors containing the nucleic acid molecules of theinvention and host cells transfected with the recombinant expressionvectors of the invention.

The invention provides a method for identifying agents which canupregulate or downregulate expression of pp32 in cells. Such agents canbe used to modulate the expression of pp32 in cells. Other agentsprovided by the invention which can be used to modulate the expressionand/or activity of pp32 in cells include antisense nucleic acidmolecules, ribozymes and antibodies directed against pp32. The proteinsand nucleic acids of the invention can further be used to identify andisolate proteins which interact with pp32 and map regions of interactionbetween pp32 and pp32-interactive proteins.

Compositions containing a therapeutically effective dose of pp32 orderivatives thereof in a pharmaceutically acceptable vehicle may beadministered to patients for the treatment of diseases of the immunesystem such as rheumatoid arthritis, multiple sclerosis, diabetes,morbus crohn, systemic lupus erythematosus, graft rejection andallergies. Also, these proteins may be used to identify compounds whichbind thereto, including compounds which bind and interfere with orprevent T cell activation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Immunoprecipitation of CD45 associated pp32 FIG. 1. Lane A showsan immunoprecipitation experiment with anti-CD45 antibodies using cellsthat had been lysed with TRITON X-100. Four different CD45 bands withapparent molecular weights of 220, 205, 190, and 180 kD are visible. Inaddition, there is a faint band at 32 kD. Lane B shows the same CD45immunoprecipitation obtained from cells solubilized with 1% digitonin.The 32kd band is much more pronounced under these lysis conditions. LaneA: CD45/1% Triton X-100; Lane B: CD45/1% digitonin.

FIG. 2: Determination of the isoelectric point of pp32 by twodimensional gel electrophoresis FIG. 2 shows pp32 in the two dimensionalgel electrophoresis with a molecular weight of 32 kD and an isoelectricpoint of 4.0-4.5. At the upper end of the gel the CD45 molecule can beseen with a pI of about 5.5-6. The relatively faint spot at about 56kDmost likely represents the protein kinase p56 lck.

FIG. 3A: In vitro phosphorylation of pp32 Lane A: sepharose alone; LaneB: sepharose coupled with an anti-CD3 mAb (OKT3; Ortho); Lane C:sepharose coupled with an anti-CD45 mAb (Gap 8.3 from hybridoma ATCC HB12); Lane D: sepharose coupled with anti-CD4 mAb (OKT4, Ortho).

FIG. 3B: Lanes A and B: anti-CD45 mAb (Gap 8.3); Lanes C and D:sepharose coupled with CD45RA mAb 2H4 (Coulter) that reacts with the220kD isoform of CD45; Lanes E and F: sepharose coupled with UCHL-1 mAb(DAKO) that reacts with the 180 kD isoform of CD45. Lanes A, C, E:immunoprecipitates from T cells that do not express the 180 kD isoformof CD45. Lanes B, D, F: immunoprecipitates from T cells that do notexpress the 220 kD isoform of CD45.

FIG. 4:CD45 immunoprecipitation from different cell lines FIG. 4 showsthat in vitro phosphorylation of pp32 can be observed in the cell linesJurkat and Laz-509 but not in t tPB-ALL and K562 as the latter cells donot express CD45. This suggests that the expression of CD45 is aprerequisite for the coprecipitation and subsequent in vitrophosphorylation of pp32. Lane D: Jurkat cells; Lane C: Laz-509 cells;Lane B: HPBALL cells; Lane A: K562 cells.

FIG. 5: Phosphoaminoacid analysis of in vivo and in vitro phosphorylatedpp32. A: in vivo phosphorylated pp32 from resting T cells; B: in vitrophosphorylated pp32.

FIG. 6: Characterization of the CD45 associated tyrosine kinase A: invitro phosphorylation pattern of CD4 and CD45 immunoprecipitatesobtained from T cells lysed with 1% Brij 58 (Lane A: CD45immunoprecipitate; Lane B: CD4 immunoprecipitate. B: V8 protease digestof the 56kD doublet band from A: Lane A and B as above.

FIG. 7: In vitro dephosphorylation of pp32 by CD45 Lane A: in vitrophosphorylated pp32 incubated with purified CD3 in the presence of DTT;Lane B: In vitro phosphorylated pp32 incubated with inactive CD45; LaneC: in vitro phosphorylated pp32 incubated with CD45 that had beenactivated with DTT; Lane D: in vitro phosphorylated pp32 incubated withDTT-activated CD45 in the presence of sodium-orthovanadate (10 mM,Merck).

FIG. 8: Changes in in vitro labeled pp32 in the course of T cellactivation Lane A: pp32, unstimulated T lymphocytes; Lane B: pp32; Tcells stimulated for 5 minutes with PMA; Lane C: pp32; T cellsstimulated for 10 minutes with PMA; Lane D: T cells stimulated for 30minutes with PMA; Lane E: T cells stimulated for one hour with PMA.

DETAILED DESCRIPTION OF THE INVENTION

The protein pp32 was detected by means of immunoprecipitation. Tlymphocytes from peripheral blood (European J. of Immunology, 19, p.337(1989)) were cultured for 12 hours (at 37° C. and 100% humidity) inphosphate free medium that had been supplemented with 32porthophosphate. This treatment exchanges inorganic nonradioactivephosphate for radioactive phosphate in the intracellular pool. The cellswere subsequently lysed at 4° C. for 45 minutes and the cell nucleiremoved by centrifugation. Lysates were preabsorbed for 30 minutes withCNBr activated protein A sepharose to which an irrelevant monoclonalantibody (IgGl; 6 μg/ml) had been bound. It was followed by theimmunoprecipitation with an anti-CD45 monoclonal antibody (Gap 8.3 fromhybridoma HB 12, ATCC) and the precipitate was separated by means ofelectrophoresis. It was shown that the pp32 specifically coprecipitateswith the anti-CD45 monoclonal antibody. Neither the isotype of themonoclonal antibody nor the isoform of the CD45 that is being recognizedseems to play a role. The method for kinasing the sample in vitro issimilar to the technique for immunoprecipitation of in vivo labeledmaterial.

To determine the isoelectric point, the precipitates were separated fromthe sepharose beads by means of electroelution. Subsequently the elutedproteins were precipitated with acetone. The acetone was evaporated andthe proteins were washed and dried. The pI determination was performedby two dimensional gel electrophoresis according to O'Farrel. Pp32 wasshown to have an isoelectric point of 4.0 to 4.5.

For the phosphoamino acid analysis, CD45 immunoprecipitates that hadbeen obtained from digitonin lysed T lymphocytes were separatedelectrophoretically and the location of pp32 was determined by means ofautoradiography. Subsequently pp32 was cut out of the gel, rehydrated,electroeluted, precipitated with acetone, hydrolysed and dried. Thephosphoamino acid analysis was then performed by two dimensional thinlayer chromatography on cellulose plates (Merck). It was observed thatin vivo labeled pp32 from unstimulated cells was predominantlyphosphorylated on serine. In contrast, where the CD45 immunoprecipitateswere kinased in vitro the same protein was exclusively phosphorylated ontyrosine residues. This requires the presence of a tyrosine kinase asfor example p56^(lck). In vitro experiments showed that active CD45 isable to dephosphorylate in vitro labeled pp32.

The pp32 protein of this invention can be purified in larger amountsusing otherwise conventional methods. For example, approximately 10¹⁰-10¹⁵ of CD45 positive T cells, preferably human such as the Jurkat Tcell line, are produced by cell culture using conventional methods andmaterials, including a conventional culture medium. The cells are thenseparated from the culture medium by centrifugation (pelleting) or anyother convenient methods, rinsed as desired to remove residual media ormedia components, and then lysed in an appropriate volume ofLysis-buffer supplemented with 1% digitonin. The lysate is preclearedseveral times with an appropriate volume (e.g. ˜250 μl/10⁸ cells) ofpacked CNBr Sepharose beads coupled with an unrelated mAb. The materialis then absorbed with an appropriate quantity of anti-CD45 coupledbeads. The beads are washed several times in a large volume of Lysisbuffer and a portion removed for in vitro kinasing (e.g. 100 μl ofpacked beads). The kinased material is mixed with the unlabeled materialto act as a tracer for later identification. A preparative nonreducing10% SDS-PAGE gel would be run on the material to avoid contaminationwith low molecular weight proteins (e.g. light chains of theprecipitating CD45 mAb). The position of pp32 may be identified byautoradiography, the appropriate band excised from the SDS gel and theprotein electroeluted using an electroelution chamber (Schleicher andSchuell, see protocol for two dimensional gel electrophoresis). pp32 maybe precipitated with acetone and dissolved in loading buffer for twodimensional gel electrophoresis. The precipitate may then be resolved inan isoelectric focusing system separating by pI in the first dimensionand by molecular weight in the second dimension (reducing 10% SDS-PAGE).The position of pp32 may be identified by staining the gel withCoomassie blue. In addition, the position of pp32 can be checked bymeans of autoradiography. Thus the material may be obtained, purified,eluted and sequenced.

Alternatively, CD45 and the associated pp32 protein could be removedfrom the cell lysate as a complex, e.g. by immunoprecipitation orimmunoaffinity chromatography using immobilized (or immobilizable orotherwise separable) antibody against CD45. For instance, an CD45monoclonal antibody (GAP 8.3) previously bound to protein A-sepharosebeads can be used to immunoprecipitate the CD45 complex containing pp32protein. The beads would then be separated from the remaining componentsof the cell lysate and washed, e.g. in lysis buffer, to remove anyextraneous materials including non-CD45-associated constituents of thecell lysate. The separated beads bearing the pp32 protein would then betreated to release the pp32 protein which may then be separatelyrecovered. For instance, the beads may be resuspended in a TRITON X-100containing buffer to release the bound pp32 into solution.Alternatively, the beads may be treated with 6M guanidine to releasebound proteins. Whichever method is chosen, the pp32 protein may beseparated from other proteins in the solution by conventional methodssuch as reverse phase HPLC (which can be relied upon to achieve puritylevels typically in excess of 95%) or by SDS-PAGE. Such methods shouldprovide sufficiently purified samples of pp32 protein to permit furthercharacterization of the protein, including amino acid sequencing --whether pp32 was purified by HPLC or cut from a nitrocellulose membraneto which it had been blotted from a polyacrylamide gel.

The fact that this protein can now be obtained in pure form (preferablyat least about 90% free from other human proteins with which it isassociated in nature, and more preferably at least about 95% free) bythe above-described methods now makes it possible for one to applyconventional methods to elucidate the amino acid composition andsequence of the proteins as mentioned above; and to produce antisera ormonoclonal antibodies capable of specifically binding to p32, to recoverand further purify the pp32 protein, if desired, by adaptation ofotherwise conventional methods including reverse phase HPLC and/or otherchromatographic methods, including immunoaffinity techniques, forinstance using specific anti-pp32 antibodies. Such antibodies may alsobe used to immunopurify proteins associated with pp32. In such methodspp32 and associated protein(s) would be separated from other materialsby virtue of binding to the anti-pp32 antibodies (which may beimmobilized), the bound materials would then be washed to removecontaminants, i.e., materials not specifically associated with the pp32protein or with proteins specifically associated with pp32, and themolecules associated with the pp32 may then be separately released fromthe pp32, for example by altering the ionic strength of the buffer, andthen removing materials as released from the association.

This invention thus identifies for the first time the existence of anovel component of the T cell cellular machinery. Given the informationdisclosed herein, the protein can now be obtained for the first time andin purified form, e.g. from natural sources (such as cultured CD45+ Tcells). Compositions containing pp32 protein can be used to producespecific antibodies capable of recognizing and binding to p32. pp32proteins can be purified directly by immunoaffinity methods using suchantibodies rather than indirectly with CD45 antibodies as discussedpreviously. That may be especially desirable in cases where there may bea molar excess of pp32 relative to CD45, e.g. in the case ofheterologous overexpression of a DNA sequence encoding pp32 or a muteinthereof. In addition, pp32 antibodies can be used to screen or identifycells which produce higher levels of p32. Such cells, including perhapsgenetically engineered host cells which overexpress pp32 or muteinsthereof can be used to screen molecules to identify those which act asmodulators of T cell activation.

The ability to isolate pp32 protein allowed for the determination of theamino acid sequences of several peptide fragments of the protein. Thesepeptide fragments included an amino-terminal fragment, tryptic fragmentsand V8 protease fragments. Based upon the amino acid sequences of thesepeptide fragments, degenerate oligonucleotide primers could be designedwhich are complementary to nucleic acid encoding pp32. These primerswere used in polymerase chain reactions to amplify small fragments ofthe pp32 cDNA. A small pp32 cDNA fragment was then used a probe toscreen a human cDNA library to obtain a full-length cDNA encoding pp32.The nucleotide sequence of a pp32 cDNA is shown in SEQ ID NO: 1. Thepredicted amino acid sequence of the protein encoded by this cDNA isshown in SEQ ID NO: 2. In vitro translation of the isolated cDNAproduced a protein which migrated in polyacrylamide gels at a molecularweight of 32 kD. The in vitro translated protein was immunreactive withan antiserum raised against isolated pp32 protein from Jurkat cells. Theamino acid sequence of the amino-terminal peptide fragment of pp32begins at amino acid position 21 of SEQ ID NO: 2, which likely indicatesthat the first 20 amino acids of the predicted protein correspond to asignal sequence. Furthermore, there is a stretch of hydrophobic aminoacid residues close to the N-terminal end of the mature protein,consistent with pp32 being a membrane-bound protein.

The invention provides an isolated nucleic acid molecule encoding a pp32protein which associates with CD45. The term "isolated" as used hereinrefers to a nucleic acid molecule substantially free of cellularmaterial or culture medium when produced by recombinant DNA techniques,or chemical precursors or other chemicals when chemically synthesized.An "isolated" nucleic acid molecule is also free of sequences whichnaturally flank the nucleic acid (i.e., sequences located at the 5' and3' ends of the nucleic acid) in the organism from which the nucleic acidis derived. The term "nucleic acid" is intended to include DNA and RNAand can be either double stranded or single stranded. In one embodiment,the isolated nucleic acid molecule is a cDNA. Preferably, the pp32protein encoded by the nucleic acid is a human protein. In oneembodiment, the nucleic acid encoding a human pp32 protein comprises anucleotide sequence shown in SEQ ID NO: 1. In another embodiment, thenucleic acid encoding a human pp32 protein comprises a coding region ofthe nucleotide sequence shown in SEQ ID NO: 1.

An isolated nucleic acid of the invention can be isolated using standardmolecular biology techniques. For example, a nucleic acid moleculeencoding a pp32 protein can be amplified from genomic DNA or cDNA by thepolymerase chain reaction using oligonucleotide primers designed basedupon the nucleotide sequence shown in SEQ ID NO: 1. Alternatively, anucleic acid molecule encoding a pp32 protein can be isolated byscreening a cDNA or genomic DNA library with a probe containing all orpart of the nucleotide sequence shown in SEQ ID NO: 1. A nucleic acid ofthe invention (for instance an oligonucleotide) can also be chemicallysynthesized using standard techniques. Various methods of chemicallysynthesizing polydeoxynucleotides are known, including solid-phasesynthesis which, like peptide synthesis, has been fully automated incommercially available DNA synthesizers (See e.g., Itakura et al. U.S.Pat. No. 4,598,049; Caruthers et al. U.S. Pat. No. 4,458,066; andItakura U.S. Pat. Nos. 4,401,796 and 4,373,071).

It will be appreciated by those skilled in the art that nucleic acidsencoding a pp32 protein which associates with CD45 which have anucleotide sequences other than those provided by the invention can beisolated or synthesized by standard techniques. For example, an isolatednucleic acid encoding a pp32 protein can have a different nucleotidesequence than that described herein due to degeneracy in the geneticcode. Such nucleic acids encode functionally equivalent proteins butdiffer in sequence from the sequences described herein due to degeneracyin the genetic code. For example, a number of amino acids are designatedby more than one triplet. Codons that specify the same amino acid, orsynonyms (for example, CAU and CAC are synonyms for histidine) may occurdue to degeneracy in the genetic code. As one example, DNA sequencepolymorphisms within the nucleotide sequence of a pp32 protein(especially those within the third base of a codon) may result in"silent" mutations in the DNA which do not affect the amino acidencoded. Isolated nucleic acids encoding a human pp32 protein having anucleotide sequence which differs from that provided herein (i.e., SEQID NO: 1) due to degeneracy of the genetic code are considered to bewithin the scope of the invention. Furthermore, it is expected that DNAsequence polymorphisms that do lead to changes in the amino acidsequences of a pp32 protein will exist within a population. It will beappreciated by one skilled in the art that these variations in one ormore nucleotides (up to about 3-4% of the nucleotides) of the nucleicacids encoding proteins having the properties of pp32 (e.g., ability toassociate with CD45) may exist among individuals within a population dueto natural allelic variation. Any and all such nucleotide variations andresulting amino acid polymorphisms are within the scope of theinvention.

The isolated nucleic acids of the invention are useful for constructingnucleotide probes for use in the detection of nucleotide sequences inbiological materials, such as cell extracts, or directly in cells (e.g.,by in situ hybridization). A nucleotide probe can be labeled with aradioactive element which provides for an adequate signal as a means fordetection and has sufficient half-life to be useful for detection, suchas ³² p, ³ H, ¹⁴ _(C) or the like. Other materials which can be used tolabel the probe include antigens that are recognized by a specificlabeled antibody, fluorescent compounds, enzymes and chemiluminescentcompounds. An appropriate label can be selected with regard to the rateof hybridization and binding of the probe to the nucleotide sequence tobe detected and the amount of nucleotide available for hybridization.The isolated nucleic acids of the invention, or oligonucleotidefragments thereof, can be used as suitable probes for a variety ofhybridization procedures well known to those skilled in the art. Theisolated nucleic acids of the invention enable one to determine whethera cell expresses an mRNA transcript encoding a pp32 protein. Forexample, mRNA can be prepared from a sample of cells to be examined andthe mRNA can be hybridized to an isolated nucleic acid encompassing anucleotide sequence comprising all or a portion of SEQ ID NO: 1.Furthermore, the isolated nucleic acids of the invention can be used todesign oligonucleotide primers, e.g. PCR primers, which allow one todetect the expression of a pp32 mRNA transcript in a cell.

The isolated nucleic acid molecules of the invention can be used inassays to screen for agents which upregulate or downregulate expressionof pp32 mRNA transcripts. The invention provides a method foridentifying an agent which can upregulate or downregulate expression ofa pp32 mRNA. The method involves contacting a cell which expresses orcan be induced to express pp32 with an agent to be tested and detectingexpression of pp32 mRNA in the cell in the presence and absence of theagent. A preferred cell type for use in the method of the invention is aT lymphocyte. The term "upregulates" encompasses inducing the expressionof pp32 mRNA in a cell which does not constitutively express pp32 orincreasing the level of expression of pp32 mRNA in a cell which alreadyexpresses pp32. The term "downregulates" encompasses decreasing oreliminating expression of pp32 mRNA in a cell which already expressespp32. The term "agent" is intended to include molecules which trigger anupregulatory or downregulatory response in a cell. For example, an agentcan be a small organic molecule, a biological response modifier (e.g., acytokine) or a molecule which can crosslink surface structures on thecell (e.g., an antibody). Expression of pp32 mRNA in a cell can bedetected, for example, by reverse transcribing mRNA from the cell andusing the cDNA thus obtained as a template in PCR reactions utilizingPCR primers which can detect pp32 cDNA (i.e., PCR primers designed basedupon the nucleotide sequence of a pp32 cDNA, e.g., SEQ ID NO: 1).Alternatively, pp32 mRNA can be detected by standard hybridizationtechniques (e.g., Northern hybridization; RNase protection) using probesencompassing all or part of a nucleotide sequence encoding a pp32protein (e.g., all or part of SEQ ID NO: 1 ). An agent which upregulatesor downregulates expression of a pp32 mRNA in a cell, identified by themethod of the invention, can be used to modulate the level of expressionof pp32 in a cell.

Another type of agent which can be used to modulate the expression ofpp32 in a cell is an antisense nucleic acid molecule. An antisensenucleic acid molecule which is complementary to a nucleic acid moleculeencoding pp32 can be designed based upon the isolated nucleic acidmolecules encoding pp32 provided by the invention. An antisense nucleicacid molecule can comprise a nucleotide sequence which is complementaryto a coding strand of a nucleic acid, e.g. complementary to an mRNAsequence, constructed according to the rules of Watson and Crick basepairing, and can hydrogen bond to the coding strand of the nucleic acid.The antisense sequence complementary to a sequence of an mRNA can becomplementary to a sequence/bund in the coding region of the mRNA or canbe complementary to a 5' or 3' untranslated region of the mRNA.Furthermore, an antisense nucleic acid can be complementary in sequenceto a regulatory region of the gene encoding the mRNA, for instance atranscription initiation sequence or regulatory element. Preferably, anantisense nucleic acid complementary to a region preceding or spanningthe initiation codon or in the 3' untranslated region of an mRNA isused. An antisense nucleic acid can be designed based upon thenucleotide sequence shown in SEQ ID NO: 1. A nucleic acid is designedwhich has a sequence complementary to a sequence of the coding oruntranslated region of the shown nucleic acid. Alternatively, anantisense nucleic acid can be designed based upon sequences of a pp32gene, which can be identified by screening a genomic DNA library with anisolated nucleic acid of the invention. For example, the sequence of animportant regulatory element can be determined by standard techniquesand a sequence which is antisense to the regulatory element can bedesigned.

The antisense nucleic acids and oligonucleotides of the invention can beconstructed using chemical synthesis and enzymatic ligation reactionsusing procedures known in the art. The antisense nucleic acid oroligonucleotide can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids e.g. phosphorothioate derivatives and acridine substitutednucleotides can be used. Alternatively, the antisense nucleic acids andoligonucleotides can be produced biologically using an expression vectorinto which a nucleic acid has been subcloned in an antisense orientation(i.e. nucleic acid transcribed from the inserted nucleic acid will be ofan antisense orientation to a target nucleic acid of interest). Theantisense expression vector is introduced into cells in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub, H. etal., Antisense RNA as a molecular tool for genetic analysis, Reviews --Trends in Genetics, Vol. 1 (1)1986

The nucleic acids of the invention can further be used to designribozymes which are capable of cleaving a single-stranded nucleic acidencoding a pp32 protein, such as a pp32 mRNA transcript. A catalytic RNA(ribozyme) having ribonuclease activity can be designed which hasspecificity for an mRNA encoding pp32 based upon the sequence of anucleic acid of the invention (e.g., SEQ ID NO: 1 ). For example, aderivative of a Tetrahymena L-19 IVS RNA can be constructed in which thebase sequence of the active site is complementary to the base sequenceto be cleaved in a pp32-encoding mRNA. See for example Cech et al. U.S.Pat. No. 4,987,071; Cech et al. U.S. Pat. No. 5,116,742. Alternatively,a nucleic acid of the invention could be used to select a catalytic RNAhaving a specific ribonuclease activity from a pool of RNA molecules.See for example Bartel, D. and Szostak, J. W. Science 261,1411-1418(1993).

An isolated nucleic acid molecule of the invention encoding a pp32protein can be incorporated in a known manner into a recombinantexpression vector which ensures good expression of the encoded proteinor portion thereof. A recombinant expression vector is suitable fortransformation of a host cell, which means that the recombinantexpression vector contains a nucleic acid or an oligonucleotide fragmentthereof of the invention and a regulatory sequence, selected on thebasis of the host cells to be used for expression, which is operativelylinked to the nucleic acid or oligonucleotide fragment. Operativelylinked is intended to mean that the nucleic acid is linked to aregulatory sequence in a manner which allows expression of the nucleicacid. Regulatory sequences are art-recognized and are selected to directexpression of the desired protein in an appropriate host cell.Accordingly, the term regulatory sequence includes promoters enhancersand other expression control elements. Such regulatory sequences areknown to those skilled in the art or one described in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, CA (1990) can be used. It should be understood that the design ofthe expression vector may depend on such factors as the choice of thehost cell to be transfected and/or the type of protein desired to beexpressed. Such expression vectors can be used to transfect cells tothereby produce proteins or peptides encoded by nucleic acids asdescribed herein.

The recombinant expression vectors of the invention can be designed forexpression of encoded proteins in prokaryotic or eukaryotic cells. Forexample, proteins can be expressed in bacterial cells such as E. coli,insect cells (using baculovirus), yeast cells or mammalian cells. Othersuitable host cells can be found in Goeddel, Gene Expression Technology:Methods in Enzyrnology 185, Academic Press, San Diego, CA (1990).

Expression in prokaryotes is most often carried out in E. coli withvectors containing constitutive or inducible promotors directing theexpression of either fusion or non-fusion proteins. Fusion vectors add anumber of amino acids usually to the amino terminus of the expressedtarget gene. Such fusion vectors typically serve three purposes: 1) toincrease expression of recombinant protein; 2) to increase thesolubility of the target recombinant protein; and 3) to aid in thepurification of the target recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the target recombinant protein to enable separation of thetarget recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne,Australia), pMAL (New England Biolabs, Beverly, MA) and pRIT5(Pharmacia, Piscataway, NJ) which fuse glutathione S-tranferase, maltoseE binding protein, or protein A, respectively, to the target recombinantprotein.

Inducible non-fusion expression vectors include pTrc (Antann et al.,(1988) Gene 69:301-315) and the pET series of vectors (Studier et al.,Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, California (1990) 60-89). In pTrc, target gene expressionrelies on host RNA polymerase transcription from a hybrid trp-lac fusionpromoter. In pET vectors, expression of inserted target genes relies ontranscription from the T7 gn10-lac 0 fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gnl ). This viral polymerase issupplied by host strains BL2 I(DE3) or HMS174(DE3) front a resident λprophage harboring a T7 gnl under the transcriptional control of thelacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector (e.g. a nucleic acid encoding a pp32 protein) so that theindividual codons for each amino acid would be those preferentiallyutilized in highly expressed E. coli proteins (Wada et al., (1992) Nuc.Acids Res. 90:2111-2118). Such alteration of nucleic acid sequences ofthe invention could be carried out by standard DNA synthesis techniques.

Examples of vectors for expression in yeast S. cerivisae includepYepSecl (Baldari. et al., (1987) Embo J. 6:229-234), pMFa (Kurjan andHerskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.).

Baculovirus vectors available for expression of proteins in culturedinsect cells (SF 9 cells) include the pAc series (Smith etal., (1983)Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow, V. A., andSummers, M. D., (1989) Virology 170:31-39).

Expression of a pp32 protein in mammalian cells is accomplished using amammalian expression vector. Examples of mammalian expression vectorsinclude pCDM8 (Seed, B., (1987) Nature 329:840) and pMT2PC (Kaufman etal. (1987), EMBO J 6:187-195). When used in mammalian cells, theexpression vector's control functions are often provided by viralmaterial. For example, commonly used promoters are derived from polyoma,Adenovirus 2, cytomegalovirus and most frequently, Simian Virus 40. Inone embodiment, the recombinant expression vector is capable ofdirecting expression of the nucleic acid preferentially in a particularcell type. This means that the expression vector's control functions areprovided by regulatory sequences which allow for preferential expressionof a nucleic acid contained in the vector in a particular cell type,thereby allowing for tissue or cell-type specific expression of anencoded protein.

The recombinant expression vector of the invention can be a plasmid.Alternatively, the recombinant expression vector of the invention can bea virus, or portion thereof, which allows for expression of a nucleicacid introduced into the viral nucleic acid. For example, replicationdefective retroviruses, adenoviruses and adeno-associated viruses can beused.

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression, by transcription of the DNA molecule, of an RNA moleculewhich is antisense to the nucleotide sequence of SEQ ID NO: 1.Regulatory sequences operatively linked to the antisense nucleic acidcan be chosen which direct the continuous expression of the antisenseRNA molecule in a variety of cell types, for instance a viral promoterand/or enhancer, or regulatory sequences can be chosen which directtissue or cell type specific expression of antisense RNA.

The recombinant expression vectors of the invention can be used to makea transformant host cell including the recombinant expression vector.The term "transformant host cell" is intended to include prokaryotic andeukaryotic cell which have been transformed or transfected with arecombinant expression vector of the invention. The terms "transformedwith", "transfected with", "transformation" and "transfection" areintended to encompass introduction of nucleic acid (e.g. a vector) intoa cell by one of many possible techniques known in the art. Prokaryoticcells can be transformed with nucleic acid by, for example,electroporation or calcium-chloride mediated transformation. Nucleicacid can be introduced into mammalian cells via conventional techniquessuch as calcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofectin, electroporation ormicroinjection. Suitable methods for transforming and transfecting hostcells can be found in Sambrook et al. (Molecular Cloning: A LaboratoryManual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), andother laboratory textbooks.

The number of host cells transfected with a recombinant expressionvector of the invention by techniques such as those described above willdepend upon the type of recombinant expression vector used and the typeof transfection technique used. Typically, plasmid vectors introducedinto mammalian cells are integrated into host cell DNA at only a lowfrequency. In order to identify these integrants, a gene that contains aselectable marker (i.e., resistance to antibiotics) can be introducedinto the host cells along with the gene of interest. Preferredselectable markers include those which confer resistance to certaindrugs, such as G418 and hygromycin. Selectable markers can be introducedon a separate vector (e.g., plasmid) from the nucleic acid of interestor, preferably, are introduced on the same vector (e.g., plasmid). Hostcells transformed with one or more recombinant expression vectorscontaining a nucleic acid of the invention and a gene for a selectablemarker can be identified by selecting for cells using the selectablemarker. For example, if the selectable marker encoded a gene conferringneomycin resistance, transformant cells can be selected with G418. Cellsthat have incorporated the selectable marker gene will survive, whilethe other cells die.

The invention provides an isolated pp32 protein which can associate withCD45. The term "isolated" refers to a protein substantially free ofcellular material or culture medium when produced by recombinant DNAtechniques, or chemical precursors or other chemicals when chemicallysynthesized. In one embodiment the pp32 protein is a human protein. Whenexpressed in a non-human cell, e.g., using a recombinant expressionvector introduced onto a non-human cell an isolated human pp32 proteincan be obtained which is free of other human proteins. Preferably, ahuman pp32 protein comprises an amino acid sequence shown in SEQ ID NO:2. Proteins which have substantial sequence homology to the amino acidsequence of SEQ ID NO: 2 are also encompassed by the invention. The term"sequences having substantial sequence homology" means those amino acidsequences which have slight or inconsequential sequence variations fromthe amino acid sequence disclosed in SEQ ID NO: 2, (i.e. a protein withthe variant amino acid sequence functions in substantially the samemanner as a protein with the amino acid sequence of SEQ ID NO: 2). Thevariations may be attributable to local mutations or structuralmodifications. It is expected that substitutions or alterations can bemade in various regions of the amino acid sequence without affectingprotein function, e.g., the ability of the protein to associate withCD45 and to be a substrate for CD45. The term "pp32 protein" is intendedto include fragments, mutants or variants of native pp32 that retain theability to associate with CD45. A "fragment" of a pp32 protein isdefined as a portion of pp32 which retains the ability to associate withCD45. For example, a fragment of pp32 has fewer amino acid residues thanthe entire protein. A "mutant" is defined as a pp32 protein having astructural change which does not eliminate the ability of the protein toassociate with CD45. For example, a mutant of pp32 may have a change(e.g., subsitution, deletion or addition) in one or more amino acidresidues of the protein. A "variant" is defined as a pp32 protein havinga modification which does not affect the ability of the protein toassociate with CD45. For example, a variant of pp32 may have alteredglycosylation or may be a chimeric protein of pp32 and another protein.Additionally, immunogenic portions of pp32 proteins are within the scopeof the invention. An immunogenic portion is typically of at least abouteight amino acids in length and can be predicted using algorithms, knownin the art, which predict which regions of a protein are located on thesurface of the protein. Additionally, peptide fragments of pp32 providedby the invention (see Example 9) can be used to generate anti-peptideantibodies. For example, an N-terminal peptide fragment encompassingamino acid positions 21-30 of SEQ ID NO: 2 or an internal peptidefragment encompassing amino acid position 175-195 of SEQ ID NO: 2 can beused as an immunogenic peptide.

A pp32 protein, or isoform or portion thereof, of the invention can beisolated by expression in a suitable host cell using techniques known inthe art. Suitable host cells include prokaryotic or eukaryotic organismsor cell lines, for example, yeast, E. coli and insect cells. Therecombinant expression vectors of the invention, described above, can beused to express pp32 in a host cell in order to isolate the protein. Theinvention provides a method of preparing an isolated protein of theinvention comprising introducing into a host cell a recombinant nucleicacid encoding the protein, allowing the protein to be expressed in thehost cell and isolating the protein. Preferably, the recombinant nucleicacid is a recombinant expression vector. Proteins can be isolated from ahost cell expressing the protein according to standard procedures of theart, including ammonium sulfite precipitation, fractionation columnchromatography (e.g. ion exchange, gel filtration, electrophoresis,affinity chromatography, etc.) and ultimately, crystallization (seegenerally, "Enzyme Purification and Related Techniques", Methods inEnzymology, 22, 233-577 (1971)). Alternatively, a pp32 protein, orportion(s) thereof; of the invention can be prepared by chemicalsynthesis using techniques well known in the chemistry of proteins suchas solid phase synthesis (Merrifield, 1964, J. Am. Chem. Assoc.85:2149-2154) or synthesis in homogeneous solution (Houbenweyl, 1987,Methods of Organic Chemistry, ed. E. Wansch, Vol. 15 I and II, Thieme,Stuttgart). Depending on its method of preparation, a pp32 protein ofthe invention may be phosphorylated or not phosphorylated. For example,pp32 isolated from resting cells (e.g., resting Jurkat cells) isphosphorylated on serine. Alternatively, pp32 isolated from activatedcells, isolated by recombinant expression of the protein in aprokaryotic cell (e.g., E. coli) or chemically synthesized may not bephosphorylated.

An isolated pp32 protein (or peptide fragment thereof), obtained bypurification of the native protein, recombinant expression of theprotein or chemical synthesis, can be used to produce antibodiesdirected against the pp32 protein. For example, the protein shown in SEQID NO: 2, or an immunogenic portion thereof, can be used generateantibodies reactive with (i.e., capable of binding to) the protein.Conventional methods can be used to prepare the antibodies. For example,a mammal, (e.g., a mouse, hamster, or rabbit) can be immunized with animmunogenic form of the protein or peptide which elicits an antibodyresponse in the mammal. Techniques for conferring immunogenicity on aprotein include conjugation to carriers or other techniques well knownin the art. For example, the protein can be administered in the presenceof adjuvant. The progress of immunization can be monitored by detectionof antibody filters in plasma or serum. Standard ELISA or otherimmunoassay can be used with the immunogen as antigen to assess thelevels of antibodies. Following immunization, antisera can be obtainedand, if desired, polyclonal antibodies isolated from the sera.

In one embodiment, the antibody which binds a pp32 protein is amonoclonal antibody. To produce monoclonal antibodies, antibodyproducing cells (lymphocytes) can be harvested from an immunized animaland fused with myeloma cells by standard somatic cell fusion proceduresthus immortalizing these cells and yielding hybridoma cells. Suchtechniques are well known in the art. For example, the hybridomatechnique originally developed by Kohler and Milstein (Nature 256,495-497 (1975)) as well as other techniques such as the human B-cellhybridoma technique (Kozbor et al., Immunol. Today 4, 72 (1983)), theEBV-hybridoma technique to produce human monoclonal antibodies (Cole etal. Monoclonal Antibodies in Cancer Therapy (1985) Allen R. Bliss, Inc.,pages 77-96), and screening of combinatorial antibody libraries (Iluseet al., Science 246, 1275 (1989)). Hybridoma cells can be screenedimmunochemically for production of antibodies specifically reactive withthe protein or portion thereof and monoclonal antibodies isolated.

The term antibody as used herein is intended to include fragmentsthereof which are also specifically reactive with pp32. Antibodies canbe fragmented using conventional techniques and the fragments screenedfor utility in the same manner as described above for whole antibodies.For example, F(ab')₂ fragments can be generated by treating antibodywith pepsin. The resulting F(ab')₂ fragment can be treated to reducedisulfide bridges to produce Fab' fragments.

Chimeric and humanized antibodies are also within the scope of theinvention. It is expected that chimeric and humanized antibodies wouldbe less immunogenic in a human subject than the correspondingnon-chimeric antibody. A variety of approaches for making chimericantibodies, comprising for example a non-human variable region and ahuman constant region, have been described. See, for example, Morrisonet al., Proc. Natl. Acad. Sci. U.S.A. 81,6851 (1985); Takeda et al.,Nature 314,452(1985), Cabilly et al., U.S. Pat. No. 4,816,567; Boss etal., U.S. Pat. No. 4,816,397; Tanaguchi et al., European PatentPublication EP 171496; European Patent Publication 0173494, UnitedKingdom Patent GB 2177096B. Additonally, a chimeric antibody can befurther "humanized" such that parts of the variable regions, especiallythe conserved framework regions of the antigen-binding domain, are ofhuman origin and only the hypervariable regions are of non-human origin.Such altered immunoglobulin molecules may be made by any of severaltechniques known in the art, (e.g., Teng et al., Proc. Natl. Acad Sci.U.S.A., 80, 7308-7312 (1983); Kozbor et al., Immunology Today, 4, 7279(1983); Olsson et al., Meth. Enzymol., 92, 3-16 (1982)), and arepreferably made according to the teachings of PCT Publication WO92/06193or EP 0239400. Humanized antibodies can be commercially produced by, forexample, Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, GreatBritain.

Another method of generating specific antibodies, or antibody fragments,reactive against an alternative cytoplasmic domain of the invention isto screen phage expression libraries encoding immunoglobulin genes, orportions thereof, with a protein of the invention, or peptide fragmentthereof (e.g., with all or a portion of a protein with the amino acidsequence of SEQ ID NO: 2). For example, complete Fab fragments, V_(H)regions and V-region derivatives can be expressed in bacteria usingphage expression libraries. See for example Ward et al., Nature341,544-546: (1989); Huse et al., Science 246, 1275-1281 (1989); andMcCafferty et al. Nature 348, 552-554 (1990).

An antibody of the invention can be used to detect a pp32 protein, e.g.in cells or cell extracts or other biological preparations which cancontain pp32. An antibody can be labeled with a detectable substance toallow detection of an antibody/antigen complex. Suitable detectablesubstances with which to label an antibody include various enzymes,prosthetic groups, fluorescent materials, luminescent materials andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; and examples of suitable radioactive material include ¹²⁵ I,¹³¹ I, ³⁵ S or ³ H.

The isolated nucleic acids of the invention can further be used tocreate a non-human transgenic animal. A transgenic animal is an animalhaving cells that contain a transgene, wherein the transgene wasintroduced into the animal or an ancestor of the animal at a prenatal,e.g., an embryonic, stage. A transgene is a DNA which is integrated intothe genome of a cell from which a transgenic animal develops and whichremains in the genome of the mature animal, thereby directing theexpression of an encoded gene product in one or more cell types ortissues of the transgenic animal. Accordingly, the invention provides anon-human transgenic animal which contains cells transfected to expresspp32 protein. Preferably, the non-human animal is a mouse. A transgenicanimal can be created, for example, by introducing a nucleic acidencoding the protein (typically linked to appropriate regulatoryelements, such as a tissue-specific enhancer) into the male pronuclei ofa fertilized oocyte, e.g., by microinjection, and allowing the oocyte todevelop in a pseudopregnant female foster animal. For example, atransgenic animal (e.g., a mouse) which expresses a human pp32 proteincan be made using the isolated nucleic acid shown in SEQ ID NO: 1.Intronic sequences and polyadenylation signals can also be included inthe transgene to increase the efficiency of expression of the transgene.These isolated nucleic acids can be linked to regulatory sequences whichdirect the expression of the encoded protein in one or more particularcell types. Methods for generating transgenic animals, particularlyanimals such as mice, have become conventional in the art and aredescribed, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009 andHogan, B. et al., (1986) A Laboratory Manual, Cold Spring Harbor, NewYork, Cold Spring Harbor Laboratory. A transgenic founder animal can beused to breed additional animals carrying the transgene.

The isolated nucleic acids of the invention can further be used tocreate a non-human "knock-out" animal. The term "knock-out animal" asused herein is intended to describe an animal containing a gene whichhas been modified by homologous recombination. The homologousrecombination event may completely disrupt the gene such that afunctional gene product can no longer be produced (hence the name"knock-out") or the homologous recombination event may modify the genesuch that an altered, although still functional, gene product isproduced. For example, an isolated nucleic acids of the invention can beused to create an animal in which the gene encoding pp32 is disrupted.Preferably, the non-human animal is a mouse. To create an animal withhomologously recombined nucleic acid, a vector is prepared whichcontains the DNA which is to replace the endogenous DNA flanked by DNAhomologous to endogenous DNA (see for example Thomas, K. R. andCapecchi, M. R. (1987) Cell 51:503). The vector is introduced into anembryonal stem cell line (e.g., by electroporation) and cells in whichthe introduced DNA has homologously recombined with the endogenous DNAare selected (see for example Li, E. et al. (1992) Cell 69:915). Theselected cells are then injected into a blastocyst of an animal (e.g., amouse) to form aggregation chimeras (see for example Bradley, A. inTeratocarcinomas and Embryonic Stem Cells. A Practical Approach, E. J.Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric embryo canthen be implanted into a suitable pseudopregnant female foster animaland the embryo brought to term. Progeny harbouring the homologouslyrecombined DNA in their germ cells can be used to breed animals in whichall cells of the animal contain the homologously recombined DNA.

Besides cell/cell interaction molecules which exist at the cell surfaceof immunocompetent cells, e.g. T lymphocytes, a second set of potentialtargets for immunotherapy exist at the level of the signal transductionpathways responsible for transmission of messages from the cell membraneto the nucleus. In this regard, many transmembrane molecules expressedat the cell surface serve as receptor structures which transmit externalsignals into the genetic machinery in the cellular nucleus. Somecytosolic molecules represent components that are associated with theinner leaf of the plasma membrane and are linked to cell surfacereceptors. The former are modified in their function followingengagement of the respective receptor structure to which they arelinked. One mechanism of modulating protein function is achieved byadding or removing phosphate residues(phosphorylation/dephosphorylation) by respective enzymes, namelyprotein kinases and protein phosphatases. The initial regulation ofcellular reactions occurs at the level of this critical balance betweenphosphorylation and dephosphorylation. Therefore, it is possible toutilize such intracellular molecules as targets for immune modulation,e.g. with the goal of downregulating immune responses in human diseaseswhich are associated with or even due to enhanced reactivity of theimmune system (i.e. chronic inflammatory disease, autoimmune disease,transplant rejection, allergy). Moreover, it is possible thatabnormalities of such proteins, e.g. due to mutation or, alternatively,abnormal regulation of these molecules due to alteration in theirrespective enzymes, could lead to aberrant/malignant growth behavior.Therefore, such molecules are also potential targets for cytostatictherapy of malignant tumors.

The isolated nucleic acids and proteins of the invention can be used toidentify and isolate molecules which interact with a pp32 protein.Regions within pp32 and/or within a pp32-interactive protein which areinvolved in the interaction between the two molecules can also bemapped. For example, an isolated nucleic acid of the invention can becloned into an expression vector that can be used in an interaction trapassay such as that described in Gyuris, J. et al. (1993) Cell 791-803. App32 protein can be used as "bait" to select other proteins whichinteract with pp32 from an expression library. Additionally, a similarassay system can be used to map regions within pp32 important forinteractions with known pp32-interactive proteins (e.g. CD45, p56lck) orto be identified pp32-interactive proteins. Likewise, regions in otherproteins important for interaction with pp32 can be so mapped. Regionsso identified can be mutated or targeted with an inhibitory agent (e.g.,antibody or peptide) to disrupt interactions between pp32 andpp32-interactive proteins.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references andpublished patents and patent applications cited throughout theapplication are hereby incorporated by reference.

Example 1. Immunoprecipitation of CD45 Associated pp32

4×10⁷ resting human T cells were washed two times in phosphate freemedium (distilled water, 2.5% Idepes-Buffer (GIBCO), 2% nonessentialamino acids (GIBCO), 1% sodium pyruvate (GIBCO), 0.25% NaHCO₃ (MERCK),1% vitamins (GIBCO), 1% Penicillin-Streptomycin (GIBCO), 0.2% Gentamycin(SIGMA), 3.5×10⁻⁴ % 2-Mercaptoethanol (SIGMA), 0.2% Bovine serum albumin(SIGMA), 1.8 mM CaCl₂, 50 mM KCl, 0.8mM MgSO₄, 0.1 M NaCl, 10 mMGlucose, 50 μM Phenol Red). Subsequently the cells were incubated for 12hours in phosphate-free medium supplemented with 1 mCi 32P PO₄ (AMERSItAM) at 37° C. and 100% humidity in sterile flasks (GREINER). After 12hours, the cells were harvested from the flasks, and washed twice in PBS(SEROMED). The cells were then lysed in 1.6 ml lysis buffer (20 mMTRIS-HCl, pH 7.5 (MERCK), 150 mM NaCl (MERCK), 10 mM NaF, 1 mM EDTA(MERCK), 10 μg/ml Aprotinin (SIGMA), 50 μ g/ml Leupeptin (SIGMA), 1 mMPMSF (SIGMA). 1 mM Na-Vanadate (MERCK)) for 45 minutes at 4° C. Asdetergent, 1% TRITON X-100 (PERCE) or 1% digitonin (SIGMA) was added tothe lysis buffer. After solubilization, the nuclei were spun out at 4°C. in an Eppendorf centrifuge at 13,000 RPMs for 15 minutes. Thepostnuclear lysates were then preabsorbed 2 times with 25 μl of anirrelevant monoclonal antibody (IgGl, 6 mg/ml) coupled to CNBr activatedprotein A sepharose for 30 minutes. The sepharose beads were spun downand replaced with fresh beads. The lysates were then incubated for 1hour at 4° C. with an anti-CD45 monoclonal antibody (Gap 8.3 fromhybridoma HB12, ATCC) bound to CNBr activated sepharose beads. Thesepharose beads were subsequently spun down again, and washed 3 timeswith lysis buffer. They were then resuspended in 60 μl SDS sample buffer(10 % Glycerol (ROTI t), 3% SDS (SERVA), 0.625 M TRIS-tHCl, pH 6.8,0.001% Bromphenol blue (SERVA)) and boiled for 5 minutes in order todissociate the antigen-antibody complex. The analysis of theimmunoprccipitated proteins was performed by means of SDS gelelectrophoresis as shown in FIG. 1 and described in the relateddescription.

Example 2. Determination of tile Isoelectric Point of pp32

Immunoprecipitates from human T lymphocytes labeled in vivo with 32PO₄were obtained as described in Example 1. The CD45 immunoprecipitateswere boiled in SDS sample buffer and subsequently separated from thesepharose beads in a biotrap electroelution chamber (SCHLEICHER &SCHUELL) in SDS running buffer (0.025 M TRIS HCl, pH 8.0 0.192 M Glycin(MERCK), 0.1% SDS) at 100 V overnight. This was followed by a bufferexchange to 15 mM ammonium bicarbonate (ROTH). Subsequently the dilutedproteins were precipitated by adding double the volume of acetone andfreezing the samples for 30 minutes at 20 ° C. using 10 μg of bovineserum albumin added previously as a carrier protein. The precipitateswere spun down (15 minutes, 10,000 RMPI, Eppendorf Centrifuge, 4° C.)and the acetone was aspirated. Remaining acetone was evaporated byplacing the sample in a Lyophylizer for 5 minutes. Finally theprecipitate was washed with 60 μl ddH₂ O and dried again for one hour.

In order to determine the isoelectric point of the immunoprecipitatedproteins, two dimensional gel electrophoresis was performed according tothe protocol of O'Farrel. The electroluted and dried proteins wereresuspended in 40 g μl of O'Farrel buffer for two dimensional gelelectrophoresis (18 % NP-40 (SERVA) 3.6 % Ampholine pH 3.5-10, 14.4%Ampholine pH 6.0-8.0 (LKB), 0.45 M Dithiothreitol (DTT, SIGMA)) andloaded on a 1 mm tube gel (pH gradient from 3.5 to 7.5 according toO'Farrel, prerun 15 minutes, 200 V, 30 min, 300 V, 30 min, 400V)). Thesample was overlaid with 10 μl 6 M urea. The tube as well as the upperelectrophoresis chamber were filled with 100 mM NaOH. The buffer for thelower chamber was a 0.085 % H₃ PO₄ solution. The isoelectric focusingprocedure was performed for 16.5 hours at 400 V and 1.5 hours at 800 V(total of 7,800 V-hours). Subsequently the two tubes were equilibratedin 0.12 M TRIS-HCl, pH 6.8, 2% SDS, 0.05 M DTT, 10% Glycerol, 0.02%Bromphenol blue according to the protocol of Lefkowitz and loaded on an18% acrylamide gel in order to separate the proteins in the seconddimension by molecular weight. The localization of the separatedproteins was determined by autoradiography. See FIG. 2 and relateddescription.

Example 3. In vitro Phosphorylation of pp32

The method used to phosphorylate proteins in vitro is similar to thatdescribed in Example 1, the difference being that the anti-CD45immunoprecipitates are obtained from T lymphocytes not previouslyincubated with radioactive orthophosphate. After the immunoprecipitationof the CD45 molecule and the subsequent washing steps, sepharose beadswere resuspended in 40 μl kinase buffer (20 mM TRIS-HCl, pH 7.5, 0.1%digitonin, 10 mM MnCl₂ (MERCK)), to which 10 μCi 32P-ATP (AMERSHAM) hadbeen added and incubated for 20 minutes at room temperature. During thekinase reaction, which requires Mn as an essential cofactor, theradioactive ATP is being cleaved by the kinase into ADP and the terminalphosphate group transferred to the substrate. The substrate can beeither an associated molecule in the immunoprecipitate or the proteinkinase itself. After 20 minutes the enzymatic reaction is stopped byadding 1 ml stopping buffer (20 mM TRIS-HCl, pH 7.5, 150 mM NaCl, 20 mMEDTA, 0.1% digitonin). The sepharose beads are subsequently washed twicewith 1 ml of stop buffer resuspended in 60 μl SDS sample buffer, boiled,and electrophoretically analyzed.

FIG. 3A shows that pp32 is only phosphorylated in vitro if an anti-CD45monoclonal antibody had been used for the immunoprecipitation. Both theisotype of the mAb and the epitope recognized by the anti-CD45 antibodyare irrelevant. On the other hand, pp32 is phosphorylated in vitro onlyif the anti-CD45 monoclonal reacts with a CD45 molecule (FIG. 3B). Thisrules out non-specific binding and phosphorylation of pp32. A monoclonalantibody directed against CD3 shows a totally different protein pattern(FIG. 3A). Therefore, coprecipitation and the in vitro phosphorylationof pp32 is dependent upon immunoprecipitation of CD45 by anti-CD45antibodies. A monoclonal antibody directed against CD4 shows a weakphosphorylation of pp32 (FIG. 3A). This phenomenon suggest that CD45 andCD4 are associated with the same set of molecules.

Example 4. Existence of pp32 in Different Cells

CD45 immunoprecipitates were performed with the following cell lines:EBV-Transformed B-cell in our case CD45RFi line Laz-509 (CD45RApositive), T-cell line Jurkat (CD45RA), T-cell line HPB-ALL (CD45negative), erythroid tumor cell line K562 (CD45 negative). 1×10⁷ cellswere lysed in digitonin lysis buffer and CD45 immunoprecipitates werepreformed as described in example 1. The samples were subsequently invitro kinased. The analysis of the phosphorylated proteins was done bymeans of SDS gel electrophoresis and autoradiography. See FIG. 4 andrelated description.

Example 5. Phosphoamino Acid Analysis

CD45 immunoprecipitates that had been obtained from digitonin lysed Tlymphocytes were separated by means of electrophoresis and the locationof pp32 was determined by autoradiography. 32p labeled pp32 was then cutout of the gel and rehydrated for 30 minutes in SDS sample buffer. Thepp32 protein was subsequently electroeluted out of the rehydrated piecesof the gel as described in example 2. After acetone precipitation theprotein was washed in 60 μl H₂ O and dried for one hour in alyophilizer. Then 100 μl of 5.7 M HCl were added to the protein pellet.The tube was subsequently incubated for an additional hour at 110° C. toallow acid hydrolysis of phosphoamino acids, and the precipitate driedovernight in a lyophilyzer. The phosphoamino acid analysis was performedby means of two dimensional thin layer chromatography on cellulose mats(MERCK). The separation of the first dimension was done for 45 minutesat 1500 V at pH 1.9 (88% formic acid: acetic acid: H₂ O = 50: 156: 1794)and in the second dimension for 30 minutes at pH 3.5(Pyridin: aceticacid: H₂ O =10:100:1890). The location of amino acid standards wasdetermined by nonhydrin staining. The location of the 32p labeled aminoacids was determined by autoradiography.

In vivo labeled pp32 obtained from resting T cells is phosphorylated onserine residues. In contrast, pp32 labeled by in vitro kinasing isexclusively phosphorylated on tyrosine. This suggests the presence of atyrosine kinase in the CD45 immunoprecipitate.

Example 6. Identification of the Tyrosine Kinase

lmmunoprecipitates from resting T cells were obtained by adding eitheranti-CD45 or anti-CD4 (OKT4, Ortho) mAbs to T cells solubilized in thedetergent Brij 58 (1% v/v, Pierce). The anti-CD4 monoclonal antibody wasused because the CD4 molecule has been shown to be associated with thetyrosine kinase p56^(lck). Subsequently, the immunoprecipitates werekinased in vitro as previously described. The analysis of thecoprecipitated proteins was done by means of autoradiography of thedried SDS gel. The bands that represent p56 lck were cut out of thedried gel and rehydrated for 15 minutes in 100 μl SDS sample buffer towhich V8 protease (SIGMA) had been added at a concentration of 100μg/ml. The rehydrated gel slices were loaded on a second SDS gel(15-22.5%, 1.5 mm thickness) and incubated for another 30 minutes. Theelectrophoresis was run at 50 V until the sample butler had completelypenetrated the stacking gel. Subsequently, the voltage was increased forfour hours to 70 V and then the power was switched off for 30 minutes.The electrophoresis was then performed under standard conditions. Afterdrying the gel, the location of the peptides was determined by means ofautoradiography.

See FIG. 6 and related description. Example A is an anti-CD45immunoprecipitate. Besides pp32, there is an in vitro labeled doublet ofapparent molecular weight 56kD. The doublet has the same electrophoreticmobility as p56^(lck) coprecipitated with the anti-CD4 mAb. These datasuggest that the proteins are identical. Further proof of their homologyis given by the V8 protease digestion in Example B. The peptide patternsof both doublets are identical. Therefore, the protein kinase thatphosphorylated pp32 in vitro is assumed to be p56^(lck).

Example 7. Dephosphorylation of pp32 by Purified CD45

As previously described, pp32 is only phosphorylated in vitro if theCD45 immunoprecipitate has been obtained from digitonin lysed cells.This argues for a weak interaction between pp32/56^(lck) and CD45. Inorder to demonstrate the dephosphorylation of pp32 by CD45, T cells werelysed in digitonin. The immunoprecipitates from digitonin lysed cellswere phosphorylated in vitro. After having stopped the reaction byaddition of EDTA the immunoprecipitates were washed and subsequentlyincubated for 10 minutes at 4° C. in 40 μl of stop buffer to which 1%Triton X-100 had been added. This treatment leads to a disruption of theCD45/pp32 complex and to the release of unbound pp32. The released pp32was then added to the CD45 immunoprecipitates that had been obtainedfrom Triton X-100 lysed T cells. Dithiothreitol (DTT, Sigma), was addedto the buffer at a final concentration of 3 μg/ml in order to activatethe CD45 molecule in vitro. In a control sample sodium orthovanadate (10mM Merck), was added to inhibit the phosphatase activity of the CD45molecule. See FIG. 7 and related description.

If CD45 is not activated by DTT, no dephosphorylation of pp32 can beobserved. If CD45 is activated by the addition of DTT, pp32 showscomplete dephosphorylation. Inhibition of activated CD45 by vanadateprevents the dephosphorylation of pp32. A control CD3 immunoprecipitatedid not show any phosphatase activity in the presence of DTT. Therefore,the in vitro dephosphorylation of pp32 is dependent upon the phosphataseactivity of CD45. This suggests that pp32 might be a substrate for CD45in vivo.

Example 8. Modifications to pp32Associated with T Cell Activation

1×10⁷ Jurkat cells (10⁷ cells/ml RMPI 1640, 10% FCS) were activated forvarious times with PMA (final concentration 10⁻⁸ M). As a control,unstimulated Jurkat were used. The cells were washed once with ice coldPBS and then lysed in 1 ml of digitonin lysis buffer. The solublefraction was immunoprecipitated with anti-CD45 mAb. Theimmunoprecipitates were washed and subsequently kinased in vitro. Afterwashing in stop buffer the samples were boiled in sample buffer. Theimmunoprecipitates were run out on an 18% acrylamide gel in order toobtain better resolution of the 32 kD range.

See FIG. 8 and related description. On an 18% SDS gel, in vitro labeledpp32 obtained from unstimulated Jurkat can be seen as two distinctbands, approximately 2kD apart from each other. Upon stimulation of theT lymphocytes with PMA, the intensity of the upper band decreases in thesamples kinased in vitro after 5 minutes of stimulation. However, athird band located between the upper and lower ones, starts to appear.This process is time dependent and complete after about 30 minutes. Thelower band seems not to undergo any changes in the in vitrophosphorylation assay.

Example 9. Isolation of a cDNA Encoding pp32

In order to isolate a cDNA encoding pp32, the amino acid sequences ofseveral peptide fragments (from the amino terminal and from tryptic andV8 protease digestion) of pp32 were determined. These amino acidsequences are as follows:

    ______________________________________    N terminus (N):    SGGSAEDSVG             (SEQ ID NO:3)    Tryptic Fragments (T1-T4)    T1  GGYYHPAR               (SEQ ID NO:4)    T2  LLWASPP                (SEQ ID NO:5)    T3  WLQAR                  (SEQ ID NO:6)    T4  AAGGQGLHVTAL           (SEQ ID NO:7)    V8 Fragments (V1-V4)    V1  LGSTDNDLERQ            (SEQ ID NO:8)    V2  EDEQDTDYDHV            (SEQ ID NO:9)    V3  GDLVLGSPGPASAGGSAE     (SEQ ID NO:10)    V4  ALLSDLHAFAGSAAWDDSARA  (SEQ ID NO:11)    ______________________________________

The sequences of the peptide fragments were used to design sets ofdegenerate oligonucleotides representing the ambiguous DNA sequenceencoding portions of the polypeptides. The nucleotide sequences of theseprimers are shown below:

    ______________________________________    Stage 1:    21AF1 YTN TCN GAY CTN CAY GC   (SEQ ID                                   NO:12)    21AF2 YTN TCN GAY TTR CAY GC   (SEQ ID                                   NO:13)    21AF3 YTN AGY GAY CTN CAY GC   (SEQ ID                                   NO:14)    21AF4 YTN AGY GAY TTR CAY GC   (SEQ ID                                   NO:15)    21AR1 GC NCK NG3C NGA RTC RTC  (SEQ ID                                   NO:16)    21AR2 GC NCK NGC RCT RTC RTC   (SEQ ID                                   NO:17)    Stage 2:    11V8F GAR CAR GAY ACN GAY TA   (SEQ ID                                   NO:18)    21V8R TCC CAG GCA GCA GAG CCA GCA                                   (SEQ ID                                   NO:19)    ______________________________________     (Y = C and T; R = A and G; K = G and T; N = A, C, G and T).

The oligonucleotides were used in various combinations as primers in thepolymerase chain reaction (PCR) to sucessfully amplify short cDNAfragments encoding pp32. One cDNA fragment so isolated was used as aprobe to screen a cDNA library to obtain full-length cDNA clones.

Amplification of 56 bp cDNA encoding 19 amino acids of peptide V4

PolyA+ RNA was prepared from the human cell line Jurkat and was reversetranscribed into double-stranded cDNA using the RNAseH- reversetranscriptase kit and protocol supplied by Gibco. Approximately 20 ng ofthis cDNA was used as the template in 8 PCR reactions with primers asfollows: Four degenerate sense primers (designated 21AFl-21AF4; shownabove) corresponding to amino acids LSDLHA in peptide V4 (amino acidpositions 3 to 8, inclusive, of SEQ ID NO: 11 ) were each paired withtwo degenerate antisense primers (designated 21AR1 and 21 AR3; shownabove) corresponding to the sequence DDSARA in peptide V4 (amino acidpositions 16 to 21, inclusive, or SEQ ID NO: 11 ). The degenerateprimers were used at a concentration of 2 to 10 pmol per permutation per100 ul reaction. Cycling parameters were as follows: after an initial 4minute incubation at 94° C., samples were taken to 94° C. for 1 minute,ramped over 2.5 minutes to an annealing temperature of 37° C. for 2 min.and then ramped over a 2.5 minute interval to 72° C. for 2 minutes. Thiswas repeated for two more cycles followed by an additional 30-37 cyclesas above except that a 55° C. annealing temperature and minimal ramptimes were chosen. In order to generate sufficient quantities of PCRproducts for visualization by ethidium bromide staining, each of the 8reactions was reamplified using as template 0.1 ul of the primaryreaction. These secondary reactions were carried out for 25 cycles(30".94° C./1',55° C./1',72° C.). Two of the 8 PCR reactions producedbands in the corr range. In order to clone the PCR fragments ofinterest, a third amplification reaction was performed using as template0.1 ul of the 2 secondary reactions containing the product of interest.The primers used for the tertiary amplification were identical to thesecondary primers except that they were extended at the 5' end by thenucleotide sequence: 5' GAG TAG TCG AC 3' (SEQ ID NO: 20), whichgenerates a Sal I restriction site suitable for cloning. The PCRproducts of interest were thus cloned and sequenced. One clone contained22 base pairs between the primer-derived sequences which encoded thesequence FAGSAAW present in the V4 peptide (amino acid positions 9 to15, inclusive, in SEQ ID NO: 11 ).

Cloning of a 242 base pair cDNA fragment of pp32 by PCR

The actual DNA sequence encoding the FAGSAAW peptide (determined above)was used to design a non-degenerate antisense oligonucleotide primer(see FIG. 1 ) used in conjunction with a degenerate sense primer basedon the EQDTDY peptide sequence within peptide V2 (amino acid positions 3to 8, inclusive, in SEQ ID NO: 9). The PCR conditions were as above.This produced a 242 base pair cDNA fragment representing the partialsequence of pp32 and encoding peptide V3. This cDNA is designated probeA.

Cloning of full-length pp32 cDNA

Probe A was radioactively labeled according to standard methods and usedto screen a Jurkat cell cDNA library purchased from Stratagene in asolution consisting of 3.5X SSC, 1X Denhardt's solution, 0.4% SDS, 50ug/ml salmon sperm DNA and 10% dextran sulfate at 65° C. The filterswere washed in 2X SSC/0.1% SDS at 55° C. Approximately 30 positiveclones were obtained out of 700,000 plaques screened. The longest cloneswere selected for farther sequencing and analysis. The nucleotidesequence of clone 8 is shown in SEQ ID NO: 1 and the predicted aminoacid sequence of the encoded protein is shown in SEQ ID NO: 2. Theidentity of this cDNA clone as pp32 was further confirmed by anexperiment demonstrating that the clone directed the synthesis of apolypeptide in vitro, which migrated in polyacrylamide gels at 32 kD,and was immunoreactive with an anti-serum raised against Jurkatcell-derived pp32 protein.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 20    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 953 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 64..681    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    ACTTCTCGCTCGACACATCCAGAGCTGGAGGTGCGTGCCCGGCACGGAGGGGCCTGCGGA60    CCAATGGCTCTGCCCTGCACCTTAGGGCTCGGGATGCTGCTGGCCCTG108    MetAlaLeuProCysThrLeuGlyLeuGlyMetLeuLeuAlaLeu    151015    CCAGGGGCCTTGGGCTCGGGTGGCAGCGCGGAGGACAGCGTGGGCTCC156    ProGlyAlaLeuGlySerGlyGlySerAlaGluAspSerValGlySer    202530    AGCTCTGTCACCGTTGTCCTGCTGCTGCTGCTGCTCCTACTGCTGGCC204    SerSerValThrValValLeuLeuLeuLeuLeuLeuLeuLeuLeuAla    354045    ACTGGCCTAGCACTGGCCTGGCGCCGCCTCAGCCGTGACTCAGGGGGC252    ThrGlyLeuAlaLeuAlaTrpArgArgLeuSerArgAspSerGlyGly    505560    TACTACCACCCGGCCCGCCTAGGTGCCGCGCTGTGGGGCCGCACGCGG300    TyrTyrHisProAlaArgLeuGlyAlaAlaLeuTrpGlyArgThrArg    657075    CGCCTGCTCTGGGCCAGCCCCCCAGGTCGCTGGCTGCAGGCCCGAGCT348    ArgLeuLeuTrpAlaSerProProGlyArgTrpLeuGlnAlaArgAla    80859095    GAGCTGGGGTCCACAGACAATGACCTTGAGCGACAGGAGGATGAGCAG396    GluLeuGlySerThrAspAsnAspLeuGluArgGlnGluAspGluGln    100105110    GACACAGACTATGACCACGTCGCGGATGGTGGCATGCAGGCTGACCCT444    AspThrAspTyrAspHisValAlaAspGlyGlyMetGlnAlaAspPro    115120125    GGGGAAGGCGAGCAGCAATGTGGAGAGGCGTCCAGCCCAGAGCAGGTC492    GlyGluGlyGluGlnGlnCysGlyGluAlaSerSerProGluGlnVal    130135140    CCCGTGCGGGCTGAGGAAGCCAGAGACAGTGACACGGAGGGCGACCTG540    ProValArgAlaGluGluAlaArgAspSerAspThrGluGlyAspLeu    145150155    GTCCTCGGCTCCCCAGGACCAGCGAGCGCAGGGGGCAGTGCTGAGGCC588    ValLeuGlySerProGlyProAlaSerAlaGlyGlySerAlaGluAla    160165170175    CTGCTGAGTGACCTGCACGCCTTTGCTGGCAGCGCAGCCTGGGATGAC636    LeuLeuSerAspLeuHisAlaPheAlaGlySerAlaAlaTrpAspAsp    180185190    AGCGCCAGGGCAGCTGGGGGCCAGGGCCTCCATGTCACCGCACTG681    SerAlaArgAlaAlaGlyGlyGlnGlyLeuHisValThrAlaLeu    195200205    TAGAGGCCGGTCTTGGTGTCCCATCCCTGTCACAGCCGCTCACTCCCCGTGCCTCTGCTT741    CCCAAGATGCCATGGCTGGACTGGACCCCCAGCCCACATGACCATGCCTCAGACTGTCAC801    CCCTACCAGTTCCCAAGTCCATGTGTACCCCGCTCACCACGGGAACGGCCCCCCCCAACC861    ACAGGCATCAGGCAACCATTTGAAATAAAACTCCTTCAGCCTGTGAAAAAAAAAAAAAAA921    AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA953    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 206 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    MetAlaLeuProCysThrLeuGlyLeuGlyMetLeuLeuAlaLeuPro    151015    GlyAlaLeuGlySerGlyGlySerAlaGluAspSerValGlySerSer    202530    SerValThrValValLeuLeuLeuLeuLeuLeuLeuLeuLeuAlaThr    354045    GlyLeuAlaLeuAlaTrpArgArgLeuSerArgAspSerGlyGlyTyr    505560    TyrHisProAlaArgLeuGlyAlaAlaLeuTrpGlyArgThrArgArg    65707580    LeuLeuTrpAlaSerProProGlyArgTrpLeuGlnAlaArgAlaGlu    859095    LeuGlySerThrAspAsnAspLeuGluArgGlnGluAspGluGlnAsp    100105110    ThrAspTyrAspHisValAlaAspGlyGlyMetGlnAlaAspProGly    115120125    GluGlyGluGlnGlnCysGlyGluAlaSerSerProGluGlnValPro    130135140    ValArgAlaGluGluAlaArgAspSerAspThrGluGlyAspLeuVal    145150155160    LeuGlySerProGlyProAlaSerAlaGlyGlySerAlaGluAlaLeu    165170175    LeuSerAspLeuHisAlaPheAlaGlySerAlaAlaTrpAspAspSer    180185190    AlaArgAlaAlaGlyGlyGlnGlyLeuHisValThrAlaLeu    195200205    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: N-terminal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    SerGlyGlySerAlaGluAspSerValGly    1510    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 8 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    GlyGlyTyrTyrHisProAlaArg    15    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 7 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    LeuLeuTrpAlaSerProPro    15    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 5 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    TrpLeuGlnAlaArg    15    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    AlaAlaGlyGlyGlnGlyLeuHisValThrAlaLeu    1510    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    LeuGlySerThrAspAsnAspLeuGluArgGln    1510    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    GluAspGluGlnAspThrAspTyrAspHisVal    1510    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    GlyAspLeuValLeuGlySerProGlyProAlaSerAlaGlyGlySer    151015    AlaGlu    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    AlaLeuLeuSerAspLeuHisAlaPheAlaGlySerAlaAlaTrpAsp    151015    AspSerAlaArgAla    20    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: primer    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    YTNTCNGAYCTNCAYGC17    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: primer    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    YTNTCNGAYTTRCAYGC17    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: primer    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    YTNAGYGAYCTNCAYGC17    (2) INFORMATION FOR SEQ ID NO:15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: primer    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    YTNAGYGAYTTRCAYGC17    (2) INFORMATION FOR SEQ ID NO:16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: primer    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    GCNCKNGCNGARTCRTC17    (2) INFORMATION FOR SEQ ID NO:17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: primer    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    GCNCKNGCRCTRTCRTC17    (2) INFORMATION FOR SEQ ID NO:18:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: primer    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    GARCARGAYACNGAYTA17    (2) INFORMATION FOR SEQ ID NO:19:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: primer    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    TCCCAGGCAGCAGAGCCAGCA21    (2) INFORMATION FOR SEQ ID NO:20:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: primer    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    GACTAGTCGAC11

What is claimed is:
 1. An isolated pp32 protein which can associate withCD45 comprising an amino acid sequence shown in SEQ ID NO:
 2. 2. Theisolated pp32 protein of claim 1 which is human.
 3. The isolated pp32protein of claim 1 which is phosphorylated.
 4. The isolated pp32 proteinof claim 1 which is not phosphorylated.